Molecular Characterization of nfsA and nfsB Genes in Furazolidone-Resistant Salmonella Spp. Isolated from Poultry Eggs

Document Type : Original Articles

Authors

Department of Microbiology, Faculty of Basic Sciences, Lahijan Branch, Islamic Azad University, Lahijan, Iran.

10.32592/ARI.2025.80.2.473

Abstract

Furazolidone (FZD), a broad-spectrum antibiotic in the nitrofuran class, is banned in many countries due to health concerns. The illegal use of FZD in poultry can lead to drug resistance in bacteria, such as Salmonella spp., which infect both poultry and humans. Contaminated eggs are a primary source of Salmonella infection. This study investigated the resistance of Salmonella isolates from eggs to FZD to gain crucial insights into the prevalence of resistant strains within the population. To this end, the susceptibility of 22 Salmonella enterica isolates from eggshells to FZD was determined using the disk diffusion and minimum inhibitory concentration methods. Then, the mutations in the nfsA and nfsB genes were examined using the polymerase chain reaction method and sequencing. Results were analyzed using GeneRunner software and BLAST online software. It was found that 27.27% and 9.09% of the isolates had high and medium resistance to FZD, respectively. The minimum inhibitory concentration results were determined to be 32 μg/ml for sensitive isolates, 256 μg/ml for intermediate isolates, and 512 μg/ml for resistant isolates. Sequencing analysis identified six insertion mutations and one transition mutation in the nfsA gene of resistant isolates, as well as one silent mutation in the nfsB gene of a sensitive isolate. The study highlights substantial resistance to FZD in Salmonella isolates from eggs, associated with mutations in the nfsA gene. These findings underscore the necessity for monitoring and managing resistance in foodborne pathogens. The significant resistance to FZD and the related mutations in the nfsA gene highlight the critical need for continuous surveillance and research to address the growing issue of antimicrobial resistance, especially in food products.

Keywords

Main Subjects

References

  1. Rana A, Prince MMB, Chakma D, Islam H, Nabi M, Saifullah A. Validation of a commercial ELISA kit for screening 3-amino-2-oxazolidinone, a furazolidone antibiotic residue in shrimp. Annual Research & Review in Biology. 2020:1-10.
  2. Nehru R, Chen C-W, Dong C-D. Recent advances in electrochemical detection of furazolidone: A review. Microchemical Journal. 2024;197:109901.
  3. Jia J, Zhang H, Qu J, Wang Y, Xu N. Immunosensor of nitrofuran antibiotics and their metabolites in animal-derived foods: A review. Frontiers in chemistry. 2022;10:813666.
  4. Castanon J. History of the use of antibiotic as growth promoters in European poultry feeds. Poultry science. 2007;86(11):2466-71.
  5. Tripathi A, Suriyamoorthy P, Rawson A. Nitrofuran residues in animal sourced food: Sample extraction and identification methods–A review. Food Chemistry Advances. 2023;3:100396.
  6. Le VVH, Rakonjac J. Nitrofurans: Revival of an “old” drug class in the fight against antibiotic resistance. PLoS pathogens. 2021;17(7):e1009663.
  7. Martínez‐Puchol S, Gomes C, Pons MJ, Ruiz‐Roldán L, Torrents De La Peña A, Ochoa TJ, et al. Development and analysis of furazolidone‐resistant E scherichia coli mutants. Apmis. 2015;123(8):676-81.
  8. Gast RK, Porter Jr RE. Salmonella infections. Diseases of poultry. 2020:717-53.
  9. Singh S, Yadav AS, Singh SM, Bharti P. Prevalence of Salmonella in chicken eggs collected from poultry farms and marketing channels and their antimicrobial resistance. Food Research International. 2010;43(8):2027-30.
  10. Gholipour-Shoshod A, Rahimi S, Salehi TZ, Torshizi MAK, Behnamifar A, Ebrahimi T, et al. Evaluating the competitiveness of medicinal plants with antibiotics to control Salmonella enterica serovar Typhimurium in broiler chickens. 2023.
  11. Haghighatnezhad H, Peighambari SM, Razmyar J. Detecting Virulence Genes Among Salmonella Serovar Infantis Isolated From Poultry Sources. Iranian Journal of Veterinary Medicine. 2023;17(4).
  12. de Oliveira SlD, Flores FS, dos Santos LR, Brandelli A. Antimicrobial resistance in Salmonella enteritidis strains isolated from broiler carcasses, food, human and poultry-related samples. International journal of food microbiology. 2005;97(3):297-305.
  13. Azizpour A. Determining the antibiotic resistance patterns of isolated Salmonella from broiler flocks to 28 antimicrobial agents used in Iran. 2018.
  14. Raeisi E, Ghiamirad M. Survey on prevalence of Salmonella serogroups and antibiotics susceptibility pattern in chicken meat in Ardabil, Iran. Journal of Ardabil University of Medical Sciences. 2015;15(3):320-9.
  15. Amiri HM, Tavakoli H, Hashemi G, Mousavi T, Rostami H, Fesharaki MG, et al. The occurrence of residues of furazolidone metabolite, 3-Amino-2-Oxazolidone, in eggs distributed in Mazandaran province, Iran. Scimetr. 2014;2(4).
  16. Fazlara A, Mayahi M, Najafzadeh Varzi H, Gudarznia F, Mohammadyari S. Determination the Amount of Illegal Furazolidone Residues in Broilers in Ahvaz Abattoir by HPLC Method. Armaghane Danesh. 2014;19(3):252-64.
  17. Alawi M. Analysis of furazolidone and furaltadone in chicken tissues and eggs using a modified HPLC/ELCD method. 2000.
  18. Chousalkar K, Flynn P, Sutherland M, Roberts JR, Cheetham BF. Recovery of Salmonella and Escherichia coli from commercial egg shells and effect of translucency on bacterial penetration in eggs. International journal of food microbiology. 2010;142(1-2):207-13.
  19. ISO E. 6579-1: 2017 Microbiology of the food chain–Horizontal method for the detection, enumeration and serotyping of Salmonella–Part 1: Detection of Salmonella spp. International Organization for Standardization: Geneva, Switzerland. 2017.
  20. Holt J, Krieg N, Sneath P, Staley J, Williams S. Determinatively anaerobic Gram-negative rods. Bergey’s manual of determinative bacteriology. 1994;9:175-289.
  21. Grimont PA, Weill F-X. Antigenic formulae of the Salmonella serovars. WHO collaborating centre for reference and research on Salmonella. 2007;9:1-166.
  22. Wayne P. Clinical and laboratory standards institute: performance standards for antimicrobial susceptibility testing: informational supplement, M100. Clinical and Laboratory Standards Institute (CLSI). 2018.
  23. Moosavy M-H, Esmaeili S, Amiri FB, Mostafavi E, Salehi TZ. Detection of Salmonella spp in commercial eggs in Iran. Iranian journal of microbiology. 2015;7(1):50.
  24. Mosleh H, Rahimi-Kakolaki M. Comparative Evaluation of Disinfectants’ Efficacy in Reducing Bacterial and Fungal Contamination in Livestock Feed Production. Iranian Journal of Veterinary Medicine. 2025;19(2):385-96.
  25. El Allaoui A, Rhazi F, Essahale A, Bouchrif B, Karraouan B, Ameur N, et al. Characterization of antimicrobial susceptibility, virulence genes and identification by 16S ribosomal RNA gene sequencing of Salmonella serovars isolated from turkey meat in Meknes, Morocco. Int J Microbiol Immunol Res. 2013;1:68-79.
  26. Castro-Vargas RE, Herrera-Sánchez MP, Rodríguez-Hernández R, Rondón-Barragán IS. Antibiotic resistance in Salmonella spp. isolated from poultry: A global overview. Veterinary world. 2020;13(10):2070.
  27. Akbarmehr J. Antimicrobial resistance in Salmonella isolated from broiler chicken carcasses. Afr J Microbiol Res. 2012;6:1485-8.
  28. Azizpour A. A Study of Salmonella Spp. Contamination Rate of Eggs and Assessment of their Antibiotic Resistance Pattern in Ardabil, Iran. Qom University of Medical Sciences Journal. 2020;14(1):38-50.
  29. Bahramianfard H, Derakhshandeh A, Naziri Z, Khaltabadi Farahani R. Prevalence, virulence factor and antimicrobial resistance analysis of Salmonella Enteritidis from poultry and egg samples in Iran. BMC veterinary research. 2021;17(1):196.
  30. Shahbazi G, Shayegh J, Ghazaei S, Movassagh Ghazani MH, Hanifian S. Prevalence, virulence factor and antibiotic susceptibility patterns of Salmonella spp. from poultry products in Ardabil province. 2022.
  31. Djeffal S, Houssou H, Roheela Y, Boucebaine I, Belmeguenai M, Bouaziz O. Assessment of Bacteria and Physicochemical Parameters in Poultry Drinking Water in Skikda Region (Algeria). 2025.
  32. Antunes P, Machado J, Peixe L. Illegal use of nitrofurans in food animals: contribution to human salmonellosis? Clinical microbiology and infection. 2006;12(11):1047-9.
  33. Zhong S, Huang H, Wang X, Yu X, Shentu X. Immunochromatographic method based on PCN-222 with dual-signal output for the rapid and sensitive detection of furazolidone metabolites in animal-derived food. LWT. 2024;198:115996.
  34. Peyghan R, Varzi HN, Jamzadeh E. Determination of Furazolidone residues in muscles of the Cultured Common Carp following experimental bath and oral administration. 2012.
  35. Jahantigh M, Jafari SM, Rashki A, Salari S. Prevalence and antibiotic resistance of Salmonella spp. in Turkey. Open Journal of Medical Microbiology. 2015;5(03):113.
  36. Sarba EJ, Kudama K, Dandecha M, Megersa L, Borena BM, Gebremdhin EZ. Prevalence, organ distribution and antimicrobial susceptibility profile of Salmonella isolated from chickens purchased from markets in selected districts of West Shoa, Ethiopia. Ethiopian Veterinary Journal. 2020;24(2):73-89.
  37. Punchihewage-Don AJ, Schwarz J, Diria A, Bowers J, Parveen S. Prevalence and antibiotic resistance of Salmonella in organic and non-organic chickens on the Eastern Shore of Maryland, USA. Frontiers in Microbiology. 2024;14:1272892.
  38. García V, Montero I, Bances M, Rodicio R, Rodicio MR. Incidence and genetic bases of nitrofurantoin resistance in clinical isolates of two successful multidrug-resistant clones of Salmonella enterica serovar typhimurium: pandemic “DT 104” and pUO-StVR2. Microbial Drug Resistance. 2017;23(4):405-12.
  39. Shanmugam D, Esak SB, Narayanaswamy A. Molecular characterisation of nfsA gene in nitrofurantoin resistant uropathogens. Journal of Clinical and Diagnostic Research: JCDR. 2016;10(6):DC05.
  40. Sandegren L, Lindqvist A, Kahlmeter G, Andersson DI. Nitrofurantoin resistance mechanism and fitness cost in Escherichia coli. Journal of Antimicrobial Chemotherapy. 2008;62(3):495-503.
  41. Wan Y, Mills E, Leung RC, Vieira A, Zhi X, Croucher NJ, et al. Alterations in chromosomal genes nfsA, nfsB, and ribE are associated with nitrofurantoin resistance in Escherichia coli from the United Kingdom. Microbial Genomics. 2021;7(12):000702.

42.       Whiteway J, Koziarz P, Veall J, Sandhu N, Kumar P, Hoecher B, et al. Oxygen-insensitive nitroreductases: analysis of the roles of nfsA and nfsB in development of resistance to 5-nitrofuran derivatives in Escherichia coli. Journal of bacteriology. 1998;180(21):5529-39.

Archives of Razi Institute
Volume 80, Issue 2
March and April 2025
Pages 473-482

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